An article by David Brown, Christa Ehmen, Angela Mika & Ana Maria Bispo de Filippis
One of the challenges for the consortium has been to identify specific and sensitive zika antibody assays for use in its prospective and retrospective studies. Recently the Diagnostic Development Laboratory of the BNITM (Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany) has developed the BLACKBOX® ZIKV IgG and IgM ELISA Kits for qualitative detection of IgG and IgM antibodies to ZIKV in human serum. Together with the laboratory of Dr. Ana Maria Bispo de Filippis, Fiocruz, Rio de Janeiro, these two assays have been validated using a large panel of sera (n=422) from PCR confirmed ZIKV and other flaviviral and non-flaviviral infections samples from Brazil, Canada, Europe and Colombia.
The BLACKBOX® ZIKV IgM and IgG ELISA Kits are based on the patented Immune Complex Binding (ICB) ELISA technology . Diluted serum samples and recombinant ZIKV antigen are co-incubated in a microwell plate coated with a recombinant IgM or IgG immune complex specific capture molecule. For the IgG assay protein competitors suppressing cross-reactivity (non-specific binding) of other flaviviral infections are added. During the incubation period of 24 h at 4°C, the immune complexes are formed and bind specifically with high affinity to the capture molecule. All antibodies not binding to the antigen and excess labeled antigen are removed in the subsequent washing step. The bound immune complexes are visualized by application of the colorimetric HRP-substrate TMB.
ZIKV-IgG assay principle:
The extensive test validation of the IgM Kit showed it has a high sensitivity (96%) for samples collected 5-15 days after onset of symptoms and a high specificity (99 %) (n=38 patients with two paired samples each and 158 negative samples). The IgG assay showed a sensitivity of 92.9 % in samples collected 12 days after onset of symptoms and a specificity of 100 % (n=154 samples of 61 patients and 268 a priori negative samples).
To support the introduction and understanding of these assays, several practical training courses have been conducted in Rio de Janeiro and Hamburg for ZIKAction consortium laboratory staff from Fiocruz, Jamaica and Haiti.
The participants were as follows:
Brazil: Flávia Levi, Marcelle Aline Santos, Everton Rodrigues de Souza, Aline da Silva Santos and Monique Lima from Fiocruz Rio de Janeiro
Jamaica: Yakima Phillips from the University of the West Indies, Kingston
Haiti: Maha El Badry and Mahbubul Alam from the University of Florida
UK: Judith Heaney from the University College London Hospitals NHS Foundation Trust
Suzannah Lant from the University of Liverpool participated on behalf of Zika PLAN
The feedback from the trainees was very positive regarding hands-on time and the assay reproducibility (inter-operator variation less than 15%). All the trainees showed interest in testing sample from their patient cohorts in the assays, especially the highly specific ZIKV-IgG assay. Fiocruz Rio de Janeiro procured IgM and IgG kits to use in the sera from Brazilian cohorts of the ZIKAction study. BNITM is looking forward to future collaborations with other European Union Zika consortia.
 Rackow A, Ehmen C, von Possel R, Medialdea-Carrera R, Brown D, Bispo de Filippis AM, Carvalho de Sequeira P, Ribeiro Nogueira RM, Halili B, Jakupi X, Berisha L, Ahmeti S, Sherifi K, Schmidt-Chanasit J, Schmitz H, Mika A, Emmerich P, Deschermeier C. (2019) Immunoglobulin-like domain of HsFcµR as a capture molecule for detection of Crimean-Congo Hemorrhagic Fever Virus- and Zika Virus-specific IgM antibodies. Clin Chem. 2019 Feb 1. https://www.ncbi.nlm.nih.gov/pubmed/30709812
 Schmitz H, Gabriel M, Emmerich P (2011) Specific detection of antibodies to different flaviviruses using a new immune complex ELISA. Med Microbiol Immunol. 200(4):233-9 https://www.ncbi.nlm.nih.gov/pubmed/30709812
Notes on the authors:
David Brown, Ana Maria Bispo de Filippis – Fundacão Oswaldo Cruz, Laboratório de Flavivírus, Rio de Janeiro, Brazil
Christa Ehmen and Angela Mika – Bernhard Nocht Institute for Tropical Medicine, Diagnostics Development Laboratory, Hamburg, Germany
People in the first photo:
From left to right:
Mr. Mahbubul Alam (PhD student working with ZIKAction in Haiti)
Dr. Christina Deschermeier (Research scientist at Nermjard-Nocht Institute for Tropical Medicine)
Ms. Christa Ehmen (Lab manager at Bernhard Nocht Institut)
Second row left to right:
Dr. Maha A. Elbadry (postdoc at UF for ZIKAction Research in Haiti).
Dr. Jude Heaney (Advanced Pathogen Diagnostic Unit APDU, University College London Hospitals).